Biotechnology and Bioengineering

The feasibility of anaerobic methanethiol (MT) degradation at elevated sodium concentrations was investigated in a mesophilic (30 degrees C) lab-scale upflow anaerobic sludge bed (UASB) reactor, inoculated with estuarine sediment originating from the Wadden Sea (The Netherlands). MT was almost completely degraded (>95%) to sulfide, methane and carbon dioxide at volumetric loading rates up to 37 mmol MT x L(-1) x day(-1), 0.5 M sodium (NaCl or NaHCO(3)) and between pH 7.3 and 8.4. Batch experiments revealed that inhibition of MT degradation started at sodium (both NaCl and NaHCO(3)) concentrations exceeding 0.8 M. Sulfide inhibited MT degradation already around 3 mM (pH 8.3).

Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. Recently, it has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a 50% higher growth rate compared to a 1 L batch cultivation [Grünberger et al. (2012) Lab Chip]. We here compare growth of C. glutamicum with glucose as substrate at different scales covering batch cultivations in the liter range down to single cell cultivations in the picoliter range. The maximum growth rate of standard batch cultures as estimated from different biomass quantification methods is ${\hat {\mu }} = 0.42\pm 0.03\,{\rm h}^{- 1} $ even for microtiter scale cultivations. In contrast, growth in a microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth rate of ${\hat {\mu }} = 0.62\pm 0.02\,{\rm h}^{- 1} $. When in the same perfusion system cell-free supernatant from exponentially grown shake flask cultures is used the growth rate of single cells is reduced to ${\hat {\mu }} = 0.47\pm 0.02\,{\rm h}^{- 1} $. Likewise, when fresh medium is additionally supplied with 5 mM acetate, a growth rate of ${\hat {\mu }} = 0.51\pm 0.01\,{\rm h}^{- 1} $ is determined. These results prove that higher growth rates of C. glutamicum than known from typical batch cultivations are possible, and that growth is definitely impaired by very low concentrations of byproducts such as acetate. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.

In a former study we showed that C. glutamicum grows much faster in defined CGXII glucose medium when growth was initiated in highly diluted environments (Grünberger et al. 2013b). Here we studied the batch growth of C. glutamicum in CGXII at a comparable low starting biomass concentration of OD≈0.005 in more detail. During bioreactor cultivations a bi-phasic growth behavior with changing growth rates was observed. Initially the culture grew with μˆ=0.61±0.02 h-1 before the growth rate dropped to μˆ=0.46±0.02 h-1. We were able to confirm the elevated growth rate for C. glutamicum in CGXII and showed for the first time a growth rate beyond 0.6 in lab-scale bioreactor cultivations on defined medium. Advanced growth studies combining well-designed bioreactor and microfluidic single-cell cultivations (MSCC) with quantitative transcriptomics, metabolomics and integrative in silico analysis revealed protocatechuic acid as a hidden co-substrate for accelerated growth within CGXII. The presented approach proves the general applicability of MSCC to investigate and validate the effect of single medium components on microorganism growth during cultivation in liquid media, and therefore might be of interest for any kind of basic growth study. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.

The initial rates of carboxybenzoyl-alanyl-l-leucyl-amide (Z-L-Ala-L-Leu-NH(2)) synthesis from carboxybenzoyl-L-alanine (Z-L-Ala) and L-leucineamide (L-Leu-NH(2)) and Z-L-Ala-L-Leu-NH(2) hydrolysis in a homogeneous dimethyl sulfoxide-aqueous buffer solution [1:1 (v/v)] system catalyzed by PST-01 protease from Pseudomonas aeruginosa were measured under a wide range of Z-L-Ala, L-Leu-NH(2) and Z-L-Ala-L-Leu-NH(2) concentrations. The initial rates of the synthetic reaction, in which Z-L-Ala-L-Leu-NH(2) was produced from Z-L-Ala and L-Leu-NH(2), were inhibited by the substrates. Furthermore, the initial rates of the synthetic reaction were not inhibited by the product Z-L-Ala-L-Leu-NH(2), and those of the hydrolytic reaction were inhibited by Z-L-Ala and L-Leu-NH(2). All the initial rate data of the synthetic and hydrolytic reactions were well correlated with the rate equation derived based on the proposed reaction scheme.

The capability of hydrogen photoproduction under high cell density conditions was examined using synchronously grown cells of nitrogen-fixing Synechococcus sp. Miami BG 043511. Optimum hydrogen yield was obtained when vessels (25 ml) contained 0.2 to 0.3 mg chlorophyll (a) in 3-mL cell suspension. During a 24-h incubation period, an initial phase of hydrogen and carbon dioxide production and a subsequent phase of carbon dioxide uptake and oxygen accumulated as major products after 24 h. after the initial 24-h. After the initial 24-h incubation, as high as 7.4 and 3.7 L (at standard condition) of hydrogen and oxygen, respectively, accumulated in vessels with 22-ml gas phase. This indicated that the pressure in the flask increased to 1.5 atmosphere. Energy conversion efficiency based on photosynthetically active radiation (25 W/m(2)) was about 2.6%. However, increased pressure somehow reduced the duration of hydrogen production. Duration of hydrogen and oxygen production was prolonged by periodical (24-h interval) gas replacement during incubation. (c) 1994 John Wiley & Sons, Inc.

Synechococus sp. strain Miami BG 043511 exhibits very high H(2) photoproduction from water, but the H(2) photoproduction capability is lost rapidly with the age of the batch culture. The decreases of the capability coincides with the decrease of cellular glucose (glycogen) content. However, H(2) photoproduction capability can be restored by the addition of organic substrates. Among 40 organic compounds tested, carbohydrates such as glucose, fructose, maltose, and sucrose were effective electron donors. Among organic acids tested, only pyruvate was an effective electron donor. Among alcohols tested, glycerol was a good electron donor. These results demonstrate that this unicellular cyanobacterium exhibits a wide substrate specificity for H(2) photoproduction but has a different substrate specificity compared to photosynthetic bacteria. The maximum rates of H(2) photoproduction from a 6-day-old batch culture with 25 mmol of pyruvate, glucose, maltose, sucrose, fructose, and glycerol were 1.11, 0.62, 0.50, 0.47, 0.30, and 0.39 micromoles per mg cell dry weight per hour respectively. Therefore, this cyanobacterium strain may have a potential significance in removing organic materials from the wastewater and simultaneously transforming them to H(2) gas, a pollution free energy. The activity of nitrogenase, which catalyzes hydrogen production, completely disappeared when intracellular glucose (glycogen) was used up, but it could be restored by the addition of organic substrates such as glucose and pyruvate. (c) 1994 John Wiley & Sons, Inc.

Batch kinetic and inhibition studies were performed for the aerobic cometabolism of 1,1,1-trichloroethane (1,1,1-TCA), 1,1-dichloroethylene (1,1-DCE), and 1,1-dichloroethane (1,1-DCA) by a butane-grown mixed culture. These chlorinated aliphatic hydrocarbons (CAHs) are often found together as cocontaminants in groundwater. The maximum degradation rates (k(max)) and half-saturation coefficients (K(s)) were determined in single compound kinetic tests. The highest k(max) was obtained for butane (2.6 micromol/mg TSS/h) followed by 1,1-DCE (1.3 micromol/mg TSS/h), 1,1-DCA (0.49 micromol/mg TSS/h), and 1,1,1-TCA (0.19 micromol/mg TSS/h), while the order of K(s) from the highest to lowest was 1,1-DCA (19 microM), butane (19 microM), 1,1,1-TCA (12 microM) and 1,1-DCE (1.5 microM). The inhibition types were determined using direct linear plots, while inhibition coefficients (K(ic) and K(iu)) were estimated by nonlinear least squares regression (NLSR) fits to the kinetic model of the identified inhibition type. Two different inhibition types were observed among the compounds. Competitive inhibition among CAHs was indicated from direct linear plots, and the CAHs also competitively inhibited butane utilization. 1,1-DCE was a stronger inhibitor than the other CAHs. Mixed inhibition of 1,1,1-TCA, 1,1-DCA, and 1,1-DCE transformations by butane was observed. Thus, both competitive and mixed inhibitions are important in cometabolism of CAHs by this butane culture. For competitive inhibition between CAHs, the ratio of the K(s) values was a reasonable indicator of competitive inhibition observed. Butane was a strong inhibitor of CAH transformation, having a much lower inhibition coefficient than the K(s) value of butane, while the CAHs were weak inhibitors of butane utilization. Model simulations of reactor systems where both the growth substrate and the CAHs are present indicate that reactor performance is significantly affected by inhibition type and inhibition coefficients. Thus, determining inhibition type and measuring inhibition coefficients is important in designing CAH treatment systems.

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients is developed and presented. The method was validated by applying it to data obtained from batch kinetics of the aerobic cometabolism of 1,1,1-trichloroethane (1,1,1-TCA) by a butane-grown mixed culture. The maximum degradation rates (kmax) and half-saturation coefficients (Ks) were independently determined in single compound tests, and compared with those obtained from inhibition tests. The inhibition type was determined using direct linear plots at various substrate and inhibitor concentrations. Kinetic parameters (kmax and Ks) and inhibition coefficients (Kic and Kiu) were determined by nonlinear least squares regression (NLSR) fits of the inhibition model determined from the direct linear plots. Initial guesses of the kinetic parameters for NLSR were determined from linearized inhibition equations that were derived from the correlations between apparent maximum degradation rates (kmaxapp) and/or the apparent half- saturation coefficient (Ksapp) and the kmax, Ks, and inhibitor concentration (IL) for each inhibition equation. Two different inhibition types were indicated from the direct linear plots: competitive inhibition of 1,1,1-TCA on butane degradation, and mixed inhibition of 1,1,1-TCA transformation by butane. Good agreement was achieved between independently measured kmax and Ks values and those obtained from both NLSR and the linearized inhibition equations. The initial guesses of all the kinetic parameters determined from linear plots were in the range of the values estimated from NLSR analysis. Overall the results show that use of the direct linear plot method to identify the inhibition type, coupled with initial guesses from linearized plots for NLSR analysis, results in an accurate method for determining inhibition types and coefficients. Detailed studies with pure cultures and purified enzymes are needed to further demonstrate the utility of this method.

A H(2)-based, denitrifying and sulfate-reducing membrane biofilm reactor (MBfR) was effective for removing 1,1,1-trichloroethane (TCA) and chloroform (CF) by reductive dechlorination. When either TCA or CF was first added to the MBfR, reductive dechlorination took place immediately and then increased over 3 weeks, suggesting enrichment for TCA- or CF-dechlorinating bacteria. Increasing the H(2) pressure increased the dechlorination rates of TCA or CF, and it also increased the rate of sulfate reduction. Increased sulfate loading allowed more sulfate reduction, and this competed with reductive dechlorination, particularly the second steps. The acceptor flux normalized by effluent concentration can be an efficient indicator to gauge the intrinsic kinetics of the MBfR biofilms for the different reduction reactions. The analysis of normalized rates showed that the kinetics for reductive-dechlorination reactions were slowed by reduced H(2) bio-availability caused by a low H(2) pressure or competition from sulfate reduction.

An anaerobic microcosm set up with aquifer material from a 1,1,2,2-tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well-known H(2)-utilizing halorespiring bacteria, Sulfurospirillum (32.4 +/- 8.6% of total bacteria) and Dehalococcoides spp. (14.8 +/- 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field.

Oxidations of alcohols by alcohol dehydrogenases often suffer from low conversions and slow reaction rates due to severe product inhibition. This can be overcome by continuous product extraction, because only the concentrations, but not the kinetic parameters, can be changed. As a consequence, it is favorable to apply a differential circulation reactor with continuous product extraction, where only a small amount of product is formed per cycle. The product is then directly extracted using a microporous hydrophobic hollow fiber membrane. This results in an increase of the relative activity of the dehydrogenase at a given conversion. The reaction investigated is the kinetic resolution of racemic 1-phenyl-1,2-ethanediol by glycerol dehydrogenase (GDH). The resulting oxidation product, 2-hydroxyacetophenone, causes a strong product inhibition. Additionally, it reacts in a chemical reaction with the cofactor lowering its active concentration. Because the GDH needs beta-nicotinamide adenine dinucleotide (NAD(+)) as a cofactor, lactate dehydrogenase is used to regenerate NAD(+) from NADH by reducing pyruvate to (L)-lactate. A conversion of 50% with respect to the racemate and an enantiomeric excess >99% of the (S)-enantiomer was reached.

The catalytic properties of bovine serum albumin (BSA) have been studied rather extensively. The protein has shown catalytic activities toward a variety of reactions in aqueous media. However, there appears to be no reports of reactions catalyzed exclusively in organic media. In this study, we report the catalytic property of BSA on the isomerization of 1,2-benzisoxazole in an organic solvent. Kinetics were performed using a continuous flow system in a spectrophotometer at 330 nm. The catalysis showed a Michaelis-Menten behavior with an estimated catalytic efficiency (k(cat)/K(M)) of 8.77 M(-1)S(-1). The influence of water content and inhibitors was also studied. The activity of BSA in acetonitrile is approximately 20% of that in water under similar conditions. Inhibition studies show that the active site may be the pyridoxal binding site involving Lys-220.

Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.

A model that was used to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures in batch reactors (Sabalowsky and Semprini (in press)) was extended here to simulate observations in continuous flow suspended and attached growth reactors. The reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in a continuous flow stirred tank reactor (CFSTR); and an attached growth recirculating packed column (RPC). A concentration-dependent toxicity model used to simulate the results of batch reactors in part I (Sabalowsky and Semprini (in press) Biotechnol Bioeng) also simulated well the observations for the CFSTR and RPC growth modes. The toxicity model incorporates cDCE and TCE toxicity coefficients that directly increase the cell decay coefficient in proportion with cDCE and TCE concentrations. Simulated estimates of the cDCE and TCE toxicity coefficients indicate reductively dechlorinating cells are most sensitive to high concentrations of cDCE and TCE in batch-fed growth, followed by CFSTR, with attached growth being least sensitive. The greater toxicity of TCE than cDCE, and ratio of the modeled toxicity coefficients, agrees with previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients.

The immobilization of dextransucrase in Ca-alginate beads relies on the close association between dextran polymer and dextransucrase. However, high amounts of dextran in the enzyme preparation drastically limit the specific activity of the immobilized enzyme (4 U/mL of alginate beads). Moreover, even in the absence of diffusion limitation at the batch conditions used, the enzyme behavior is modified by entrapment so that the dextran yield increases and the alpha-1,2 glucooligosaccharides (GOS) are produced with a lower yield (46.6% instead of 56.7%) and have a lower mean degree of polymerization than with the free dextransucrase. When the immobilized catalyst is used in a continuous reaction, the reactor flow rate necessary to obtain high conversion of the substrates is very low, leading to external diffusion resistance. As a result, dextran synthesis is even higher than in the batch reaction, and its accumulation within the alginate beads limits the operational stability of the catalyst and decreases glucooligosaccharide yield and productivity. This effect can be limited by using reactor columns with length to diameter ratio > or =20, and by optimizing the substrate concentrations in the feed solution: the best productivity obtained was 3.74 g. U(-1). h(-1), with an alpha-1,2 GOS yield of 36%.

The optimization of alpha-1,2 glucooligosaccharide (GOS) synthesis from maltose and sucrose by Leuconostoc mesenteroides NRRL B-1299 dextransucrase was achieved using experimental design and consecutive analysis of the key parameters. An increase of the pH of the reaction from 5.4 to 6.7 and of the temperature from 25 to 40 degrees C significantly favored alpha-1,2 GOS synthesis, thanks to a significant decrease of the side reactions, i.e., dextran and leucrose synthesis. These positive effects were not sufficient to compensate for the decrease of enzyme stability caused by the use of high pH and temperature. However, the critical parameters were the sucrose to maltose concentration ratio (S/M) and the total sugar concentration (TSC). Alpha1,2 GOS synthesis was favored at high S/M ratios. But using these conditions also led to an increase of side reactions which could be modulated by choosing the appropriate TSC. Finally, with S/M = 4 and TSC = 45% w/v, dextran and leucrose productions were limited and the final alpha-1,2 GOS yield reached 56.7%, the total GOS yield being 88%.

A model was developed to describe toxicity from high concentrations of chlorinated aliphatic hydrocarbons (CAHs) on reductively dechlorinating cultures under batch-growth conditions. A reductively dechlorinating anaerobic Evanite subculture (EV-cDCE) was fed trichloroethene (TCE) and excess electron donor to accumulate cis-1,2-dichloroethene (cDCE) in batch-fed reactors. A second Point Mugu (PM) culture was also studied in the cDCE accumulating batch-fed experiment, as well as in a time- and concentration-dependent cDCE exposure experiment. Both cultures accumulated cDCE to concentrations ranging from 9,000 to 12,000 microM before cDCE production from TCE ceased. Exposure to approximately 3,000 and 6,000 microM cDCE concentrations for 5 days during continuous TCE dechlorination exhibited greater loss in activity proportional to both time and concentration of exposure than simple endogenous decay. Various inhibition models were analyzed for the two cultures, including the previously proposed Haldane inhibition model and a maximum threshold inhibition model, but neither adequately fit all experimental observations. A concentration-dependent toxicity model is proposed, which simulated all the experimental observations well. The toxicity model incorporates CAH toxicity terms that directly increase the cell decay coefficient in proportion with CAH concentrations. We also consider previously proposed models relating toxicity to partitioning in the cell wall (K(M/B)), proportional to octanol-water partitioning (K(OW)) coefficients. A reanalysis of previously reported modeling of batch tests using the Haldane model of Yu and Semprini, could be fit equally well using the toxicity model presented here, combined with toxicity proportioned to cell wall partitioning. A companion paper extends the experimental analysis and our modeling approach to a completely mixed reactor and a fixed film reactor.

This work investigates the use of an oil absorber as an operational strategy in vapor phase bioreactors exposed to starvation periods, during the treatment of inhibitory pollutants. After being exposed to 1,2-dichloroethane (DCE) starvation periods, the response and stability of a combined oil-absorber-bioscrubber (OAB) system was compared to that of a bioscrubber only (BO) system. In the BO system, after a 5.2 days starvation period, the DCE removal efficiency was reduced to 12%, and 6 days were needed to recover the initial removal efficiency when the DCE feed resumed. The total organic discharged (TOD(DCE)) was 16,500 g(DCE) m(bioscrubber) (-3) after the DCE starvation. Biomass analysis performed using fluorescence in situ hybridisation (FISH) showed that the microbial activity was significantly reduced during the starvation period and that 5 days were needed to recover the initial activity, after the re-introduction of DCE. In contrast, the performance of the OAB system was stable during 5.2 days of DCE starvation. The DCE removal efficiency was not affected when the DCE feed resumed and the TOD(DCE) was significantly reduced to 2,850 g(DCE) m(bioscrubber) (-3). During starvation, the activity of the microbial culture in the OAB system showed a substantially lower decrease than in the BO system and recovered almost immediately the initial activity after the re-introduction of DCE. Additionally, a mathematical model describing the performance of the OAB system was developed. The results of this study show that the OAB system can effectively sustain the biological treatment of waste gas during starvation periods of inhibitory pollutants.

Biodehalogenation of 10(-5) M concentrations of bromotrichloromethane (BTM) and 1,2-dibromo-3-chloropropane (DBCP) was studied in static cultures of Pseudomonas putida PpG-786. The experimental cultures were prepared by growing P. putida on camphor, which is known to induce the synthesis of high concentrations of cytochrome P-450 in this bacterium. Measurements of bromide ion release were found to be approximately consistent with the amounts of halocarbon degraded. Gas chromatography/elctron capture detection (GC/ECD) measurements of hydrocarbon degradation products as a function of incubation time showed the transitory appearance of chloroform and bromodichloromethane from BTM and the transitory appearance of lower boiling but unidentified products from DBCP. The degradation of BTM to trihalomethanes and the halide ion is consistent with the enzymatic reductive dehalogenation by cytochrome P-450 reported by others. The dependence of initial conversion rates on halocarbon concentration (0.1-2 ppm) and cell mass concentration (1-28 g cell/L) was determined by measuring the decline of parent halocarbon in stirred batch cell suspensions. The rate of DBCP conversion was up to 10-fold higher than the rate of BTM conversion. When the intracellular, enzyme-catalyzed conversion BTM is analyzed by the effectiveness factor of heterogeneous catalysis, the initial conversion rates measured suggest that intrinsic enzyme kinetics, rather than halocarbon permeation of the cell membrane or other diffusive processes, is rate limiting.

Due to its availability, low-price, and high degree of reduction, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. Using the platform we have established from the identification of key pathways mediating fermentative metabolism of glycerol, this work reports the engineering of Escherichia coli for the conversion of glycerol into 1,2-propanediol (1,2-PDO). A functional 1,2-PDO pathway was engineered through a combination of overexpression of genes involved in its synthesis from the key intermediate dihydroxyacetone phosphate (DHAP) and the manipulation of the fermentative glycerol utilization pathway. The former included the overexpression of methylglyoxal synthase (mgsA), glycerol dehydrogenase (gldA), and aldehyde oxidoreductase (yqhD). Manipulation of the glycerol utilization pathway through the replacement of the native E. coli PEP-dependent dihydroxyacetone kinase (DHAK) with an ATP-dependent DHAK from C. freundii increased the availability of DHAP allowing for higher 1,2-PDO production. Analysis of the major fermentative pathways identified ethanol as a required co-product while increases in 1,2-PDO titer and yield were achieved through the disruption of the pathways for acetate and lactate production. Combination of these key metabolic manipulations resulted in an engineered E. coli strain capable of producing 5.6 g/L 1,2-PDO, at a yield of 21.3% (w/w). This strain also performed well when crude glycerol, a by-product of biodiesel production, was used as the substrate. The titer and yield achieved in this study were favorable to those obtained with the use of E. coli for the production of 1,2-PDO from common sugars.

Consortia of catalase positive bacteria consisting of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Klebsiella pneumoniae, in both the planktonic form and as biofilms, disproportionate hydrogen peroxide into oxygen and water. The biofilm, however, continued to disproportionate the hydrogen peroxide in the presence of the catalase inhibitor, 3-amino-1,2,4-triazole, while the planktonic organisms did not. While the bacterial catalase-peroxidase-dismutase system was probably responsible for the disproportionation of hydrogen peroxide in both cases, biofilms resisted inhibition of this enzyme system.

1,3-Oleoyl-2-palmitoylglycerol (OPO), an important structured triglyceride in infant nutrition, was synthesized by a two-step process in high yields and purity using sn1,3-regiospecific lipases. In the first step, tripalmitin (TP) was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn1,3-regiospecific lipases yielding the corresponding 2-monopalmitin (2-MP). The 2-MP was isolated in up to 85% yield and >95% purity by crystallization and esterified in the second step with oleic acid using the same lipases to form the structured triglyceride OPO in up to 78% yield containing 96% palmitic acid in the sn2-position. Water activity, solvent, as well as carrier for lipase immobilization strongly influenced the yield and purity of products in both steps. The best results were achieved with lipases from Rhizomucor miehei and Rhizopus delemar immobilized on EP 100 and equilibrated to a water activity of 0.43. Special emphasis was given to develop this process in solvents that are allowed to be used in foodstuffs and to perform the second step in a solvent-free system.

In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast.